The Na+,K+-ATPase is the enzymatic equivalent of the Na+ pump, which couples ATP hydrolysis to the transport of three Na+ out of, and two K+ into, the cell. This coupling converts stored chemical energy into usable electrochemical energy (in the ion gradients). In addition, this protein regulates several physiological processes (directly or indirectly), including neurotransmitter release and uptake, generation of resting membrane potential, and control of vascular and visceral muscle tone. Pump activity is regulated in vivo by insulin and thyroid hormone and by putative endogenous regulators. The pump serves as the receptor for digitalis glycoside drugs. The long-term goal of this project is to understand the coupling between ion transport and the energy released by ATP hydrolysis. Although a great deal of evidence links ion transport with ATP hydrolysis through ligand-induced conformational changes, the exact mechanism of this coupling is unclear. The present proposal describes experiments to distinguish between two alternate schemes for ion transport which propose that: (1) transport is linked directly with conformational change, and that the release of ions follows the conformational change, or (2) conformational changes alter the number of cation binding sites and their affinities, and that ion transport occurs prior to these changes. Transient state experiments, which measure rate constants for different steps in the enzyme cycle, will be used to distinguish between these two possibilities. Proposed experiments are designed to measure rate constants for each step in the reaction pathway. Specifically, these experiments will measure forward and reverse rate constants for (i) substrate binding, (ii) enzyme phosphorylation, (iii) ADP and P[i] release, (iv) conformational transitions between the phosphorylated enzyme forms, (v) charge translocation, and (vi) conformational transition between the nonphosphorylated enzyme. The effect of divalent cations, alternative substrates, and other modifications on these steps will also be examined. Techniques will include stopped-flow fluorimetry and spectrophotometry, and chemical quench. Much of the earlier work in this laboratory has used enzyme labeled with the fluorescent reporter group IAF. These studies will be extended to include other fluorescence probes, such as BIPM and FITC, TNP-analogs of nucleotide di- and tri-phosphates, and pH sensitive dyes such as BCECF and SNARF. Chemical quench experiments will use radioactive substrates (e.g. [32P]- and [3H]-ATP) to examine rates of product formation.